Role of myosin light chain kinase in increase in permeability of human pulmonary microvascular endothelial cells induced by mechanical stretch

Abstract

Objective To evaluate the role of myosin light chain kinase(MLCK)in the increase in the permeability of human pulmonary microvascular endothelial cells(HPMVECs)induced by mechanical stretch.Methods HPMVECs were cultured in vitro and then randomly divided into 3 groups(n = 4 each): mechanical stretch group(group S),ML9 treatment group(group M)and control group(group C).In group S,the monolayer cells and magnetic beads coated with an RGD peptide were incubated in serum-free MCDB131 medium for 2 h,the unbound beads were washed out,and then the cells were exposed to magnetic twisting stimulation(MTS)for 2 h(frequency 3 Hz,intensity 4.2 mT).In group M,the monolayer cells and magnetic beads coated with an RGD peptide were incubated for 2 h in serum-free MCDB131 medium in which MLCK inhibitor ML9(50μmol/L)was added,and the other procedures were the same as in group S.In group C,the cells after washing as in group S were incubated for 2 h without exposing to MTS.Monolayer permeability was detected with FITC-dextran flux in transwell model.The distribution of integrin αVβ3 and actin was detected using the immunofluorescence assay.Results Compared with group C,the permeability of HPMVECs was significantly increased in group S,while no significant change was found in group M.Compared with group S,the permeability of HPMVECs was significantly decreased in group M.In addition,actin polymerized to form stress fiber,and integrin αVβ3 clustered at the end of stress fiber in group S,while actin mainly distributed in the surrounding area of the cell membrane and integrin αVβ3 evenly distributed on the cell surface in group C and M.Conclusion The mechanism by which mechanical stretch induces the increase in the permeability of HPMVECs is related to MLCK activation-mediated stress fiber formation and integrin αVβ3 clustering. Key words: Myosin-light-chain kinase; Capillary permeability; Lung